Complement activation must be tightly regulated to ensure selective targeting of invading microorganisms and avoid self-inflicted damage. (Ricklin et al., Nat. Immunol. 11:785-797 (2010)). In the classical and lectin pathways, control is achieved through recognition-driven activation on pathogen-associated molecular patterns. Activating complexes are composed of separate recognition and protease subcomponents: in the classical pathway, C1q binds to immobilised immunoglobulins, to innate immune mediators (such as pentraxins), to prions or directly to microbial surfaces to activate associated-serine proteases C1r and C1s. (Gal et al., Mol. Immunol. 46:2745-27522 (2009)). In the lectin pathway, mannan-binding lectins (MBLs, also called mannose-binding proteins) and serum ficolins bind to carbohydrates or acetylated structures on pathogens to activate MBL-associated serine proteases (MASPs), homologues of C1r and C1s. (Gal et al., supra). Once initiated, complement elicits a myriad of downstream events, including complement-induced lysis of pathogens, attraction and activation of host phagocytes and stimulation of inflammatory and adaptive immune responses. (Ricklin et al., supra).
The lectin pathway of complement is a double-edged sword. Polymorphisms in MBL and MASPs are associated with immunodeficiency, and increased severity of inflammatory disorders, such as lupus erythematosus, cystic fibrosis and rheumatoid arthritis, confirming its key defensive roles. (Garred et al. Lancet 349:236-240 (1997); Neth et al., Lancet 358:614-618 (2001); Sumiya et al., Lancet 337:1569-1570 (1991); and Kilpatrick, Biochim. Biophys. Acta 1572:401-413 (2002)). However, it is also directly implicated in exacerbating tissue damage in ischemia reperfusion injury, so selective inhibitors will provide important therapeutic benefits. (Hart et al., J Immunol 174:6373-6380 (2005) and Walsh et al., J Immunol 175:541-546 (2005)).
Mannan-binding lectins and ficolins are archetypal pattern-recognition molecules, targeting structural arrays on the surfaces of pathogens via multiple weak contacts. They have bouquet-like structures, formed from rod-like collagen-like domains, linked at the N-termini, which splay apart to terminate in clusters of three pathogen-recognition domains, and circulate in plasma associated with three different MASPs, called -1, -2 and -3. MASPs are homodimers, comprising six modular domains of which the first three, two CUB (for complement C1r/C1s, Uegf and Bmp1) modules separated by an epidermal growth factor (EGF)-like domain, are necessary and sufficient for dimerization and binding to MBL and ficolins. (Drickamer and Taylor, Ann. Rev. Cell Biol. 9:237-264 (1993); Girija et al., J. Immunol. 179:455-462 (2007); Wallis and Drickamer, J. Biol. Chem. 274:3580-3589 (1999); Krarup et al., J. Biol. Chem. 279:47513-47519 (2004); Dahl et al., Immunity 15:127-135 (2001); Takayama et al., J. Immunol. 152:2308-2316 (1994); Thiel et al., Nature 386:506-510 (1997); and Wallis and Dodd, J. Biol. Chem. 275:30962-30969 (2000)). The latter three domains, two complement control modules (CCP) and a serine protease (SP) domain, control substrate recognition and catalysis. MASP-1 and MASP-2 are synthesized as zymogens and autoactivate following cleavage of a scissile bond at the N-terminus of the SP domain. MASP-1 cleaves factor D, an early component of the alternative pathway, as well as protease-activated receptor-4 on endothelial cells to modulate the inflammatory response. (Takahashi et al., J. Exp. Med. 207:29-37, S21-23 (2010) and Megyeri et al., J. Immunol. 183:3409-3416 (2009)). Examples of full length MASP-1 amino acid sequences are provided in Accession Nos.: N_P 071593.1 (rat, set forth herein as SEQ IQ NO: 22), NP_001870 (human isoform 1, set forth herein as SEQ ID NO: 23), and NP_624302 (human isoform 2, set forth herein as SEQ ID NO: 24). MASP-2 cleaves C4 and C2 to form the C3 convertase (C4b2b), to drive lectin pathway activation. (Chen and Wallis, J. Biol. Chem. 279:26058-26065 (2004) and Rossi et al., J. Biol. Chem. 276:40880-40887 (2001)). Examples of full length MASP-2 sequences are provided in Accession Nos.: NP_742040.1 (rat, set forth herein as SEQ ID NO: 25) and NP_006601.3 (human isoform 1, set forth herein as SEQ ID NO: 26). The physiological role of MASP-3 is not known, although it differs from MASP-1 only in its SP domain, so it may modulate the activities of the other MASPs by competing for binding sites on MBL and ficolins. (Dahl et al., supra). Examples of full length MASP-3 sequences are provided in Accession Nos.: CAD32171.1 (rat, set forth herein as SEQ ID NO: 27) and AAK84071.1 (human, set forth herein as SEQ ID NO: 28). Two truncated products MAp44 and MAp19 (also called sMAp) probably perform similar functions. (Degn et al., J. Immunol. 183:7371-7378 (2009); Iwaki et al., J. Immunol. 177:8626-8632 (2006); Skjoedt et al., J. Biol. Chem. 285:8234-8243 (2010); and Stover et al., J. Immunol. 162:3481-3490 (1999)).
Complement activating complexes assemble through multiple CUB/collagen contacts, with each MASP dimer bridging up to four separate collagen-like domains of MBLs or ficolins. (Teillet et al., J. Biol. Chem. 283:25715-25724 (2008)). Equivalent interactions mediate assembly of the C1 complex of the classical pathway of complement, but via six binding sites on C1r/C1s for C1q. (Wallis et al., Immunobiology 215:1-11 (2010)). Ca2+ plays multiple roles in all three complexes: each CUB domain possesses a single Ca2+ site, which is necessary for binding to the recognition subcomponent. (Teillet et al., supra). Separate Ca2+ sites in the EGF-like domains stabilize MASP dimers and binding between C1r and C1s. (Feinberg et al., EMBO J. 22:2348-2359 (2003)). Ca2+ is also an essential part of the sugar-binding sites of the carbohydrate-recognition domains (CRDs) of MBL, and a single Ca2+ is present in each fibrinogen-like domain of ficolins and in the globular domain of C1q. (Weis et al., Nature 360:127-134 (1992); Garlatti et al., EMBO J. 26:623-633 (2007); and Gaboriaud et al., J. Biol. Chem. 278:46974-46982 (2003)).
Despite recent advances in our understanding of complement proteins, through structural analysis of individual subcomponents, the mechanism by which these subcomponents combine to activate complement is poorly understood. (Teillet et al., J. Biol. Chem. 283:25715-25724 (2008); Feinberg et al., EMBO J. 22:2348-2359 (2003); Gregory et al., J. Biol. Chem. 279:29391-29397 (2004); Weis et al., Science 254:1608-1615 (1991); Dobo et al., J. Immunol. 183:1207-1214 (2009); and Harmat et al., J. Mol. Biol. 342:1533-1546 (2004)). To address this key question, we have determined the structure of a CUB/collagen complex, together with structures of the unbound CUB and collagen-like domains and of the CUB domain with small molecule inhibitors bound to the MBL-binding site. Overall, the structures reveal the interactions that transmit the activating signal in the lectin and classical pathways, the role of Ca2+ in binding and the structural organization of full-size MBL/MASP and ficolin/MASP complexes. Furthermore, they provide insight into the initial steps leading to complement activation.